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. 2016 Aug 3;7:1212. doi: 10.3389/fmicb.2016.01212

Figure 2.

Figure 2

Construction of replacement vectors and confirmation of ChMK1 deletion mutants. (A) Strategic map of the ChMK1 replacement construct pChMK1-3300. Thick arrows indicate the orientations of ChMK1 and hygromycin phosphotransferase (hph) gene. The hph gene cassette was cloned into the corresponding sites of vector pChMK1-3300 to replace the 1108-bp of the ChMK1 ORF. (B) Colony morphologies of CH-1, ▵ChMK1-1, ▵ChMK1-8, and ChMK1-Com grown on PDA plate at 25°C for 15 d. (C) Southern blot analysis of mutants. Genomic DNA (15 μg per lane) of CH-1 (wild-type strain), ▵ChMK1-1, ▵ChMK1-8, and ChMK1-Com (ChMK1 complementary transformant) were digested with SacI. The same filter was hybridized with a probe corresponding to the ChMK1 (P). (D) Total RNA samples isolated from mycelia of CH-1, ▵ ChMK1-1, ▵ChMK1-8, and ChMK1-Com were subjected to RT-PCR using ChMK1 gene-specific primers 4F and 4R (Table 1). The RT-PCR product is a 560 bp fragment in CH-1 and ChMK1-Com as predicted, but is missing in the deletion mutants ▵ChMK1-1, ▵ChMK1-8.